Introduction
Gastric cancer (GC) is now widely recognized as one of the most common cancer types, with the sixth highest incidence (5.6%) and causing the third most cancer deaths (7.7%) worldwide (1). As a rare subtype of GC, α-fetoprotein (AFP)-producing gastric carcinoma (AFPGC) was first described by Bourreille et al (2) in 1970. AFP is typically considered an ideal clinical serum biomarker for screening and monitoring hepatocellular carcinoma, noncancerous liver diseases, yolk sac tumors and tumors of gonadal origin (3–8). The elevation of serum AFP levels has also been observed in certain other types of cancers, including cancer of the stomach, lung, pancreas, colon and bladder (9–15). Among all these organs, the stomach is believed to be the most common site of occurrence (16–18). The proportion of AFPGC among all GC cases is controversial: Reports usually estimate it to be between 1.3–7.1%, (17,19–22), whilst certain data suggest it is as high as 15% (23).
Hepatoid adenocarcinoma of the stomach (HAS), another rarer subtype of GC, was first described by Ishikura et al (24,25) in the 1980s to describe GC cases with hepatoid features. The proportion of HAS among all GC cases was previously estimated to be between 1.7–15.0‰ (26,27).
Several factors have been noted to have a significant impact on the increased risk of developing GC, including family history, diet, alcohol consumption, smoking, H. pylori and Epstein-Barr virus infections (28). There is no further evidence indicating that specific non-genetic factors are more inclined to predispose to specific subtypes such as AFPGC or HAS.
For all subtypes of GC, surgical resection remains the primary treatment strategy, including conventional surgery and endoscopic resection for early-stage lesions. Postoperative adjuvant radiotherapy, chemotherapy and targeted therapy are also utilized as supplementary treatment modalities (29).
The present report provides a description of the clinical and pathological findings, upper gastrointestinal endoscopy and enhanced chest-abdominal computed tomography (CT) images, and the outcome of surgery for an elderly patient with GC, and AFPGC and HAS features in serum test and pathology, respectively. As there are certain contemporary inconsistencies in the definitions of AFPGC and related concepts, the present report also proposes a new classification method for relevant diseases and pertinent literature is reviewed.
Case report
A 75-year-old woman presented to the General Clinic, Yancheng No. 1 People's Hospital (Yancheng, China) in November 2023 (day 0), with a chief complaint of dizziness for 1 day and a history of polydipsia and polyuria for over a decade. The patient was admitted to the ward of the Department of General Medicine with an initial diagnosis of type 2 diabetes mellitus (T2DM) and hypertension. Routine laboratory tests on admission suggested a positive fecal occult blood (OB) test, positive serum H. pylori IgG antibodies and positive H. pylori current infection marker. The patient also had mild anemia with a blood hemoglobin level of 91 g/l (reference range, 130–175 g/l). Further gastrointestinal (GI) tumor marker tests indicated an elevated serum AFP level of 183.70 ng/ml (reference range, 0–7 ng/ml). The results of other related laboratory tests, including liver function tests, Hepatitis B indicators and other GI tumor markers, were all within normal limits. Abdominal CT also revealed no significant hepatic abnormalities. Following treatment for acid suppression and gastric protection with omeprazole and sucralfate, the fecal OB test turned negative. The patient was discharged upon their request as the symptoms had subsided.
Follow-up at 1-month post-discharge (day 27) revealed a marked increase in serum AFP to 314.15 ng/ml at Qingdun Town Healthcare Center (Yancheng, China). A total of 2 weeks later, the patient presented to the General Clinic at Yancheng No. 1 People's Hospital again and was readmitted to the ward of the Department of General Medicine in December 2023 (day 43). Further tests indicated an ulteriorly elevated serum AFP level at 397.70 ng/ml. An upper gastrointestinal endoscopy revealed irregular elevations and depressions from the esophagus (40 cm from incisors) to the cardia, covered with a white coating on the surface presumably mainly made up of necrotic tissue and mucus, with a crater-like elevated nodule, indicating the presence of ulcerative GC (Borrmann II). The lesion tissue was brittle and prone to hemorrhage (Fig. 1). The endoscopic diagnosis indicated esophageal cardia cancer. Subsequent enhanced chest-abdominal CT demonstrated thickening and enhancement of the gastric wall lateral to the lesser curvature of the cardia and gastric fundus, further clarifying the extent of the lesion (Fig. 2). Endoscopic biopsy sample collected was fixed with 4% formaldehyde solution for 6 h at 25°C. Paraffin-embedded tissue sections (4 µm) were deparaffinized with xylene and rehydrated with a series of anhydrous ethanol, 95% ethanol, 70% ethanol and PBS. For pathological examination, part of the sections were stained with hematoxylin for 3 min at 25°C and eosin for 45 sec at 25°C. For immunohistochemistry (IHC), part of the sections underwent blocking of endogenous peroxidase using 3% hydrogen peroxide for 10 min at 25°C, followed by blocking of unspecific protein binding using 5% bovine serum albumin (cat. no. GC305010; Servicebio Ltd) for 1 h at 37°C. IHC sections underwent heat-mediated antigen retrieval with sodium citrate buffer (pH=6) for 10 min at 97°C and were then incubated overnight at 4°C with AFP antibody (1:100 dilution; cat. no. RMA-1069; Maxim Biotechnologies, Ltd), hepatocyte paraffin (Hep Par) 1 antibody (1:100 dilution; cat. no. MAB-1034; Maxim Biotechnologies, Ltd), cytokeratin (CK)19 antibody (1:100 dilution; cat. no. MAB-0829; Maxim Biotechnologies, Ltd) or caudal type homeobox (CDX)2 antibody (1:100 dilution; cat. no. RMA-0631; Maxim Biotechnologies, Ltd). Subsequently, IHC sections were treated with HRP-conjugated goat anti-rabbit IgG (H+L) (1:200 dilution; cat. no. GB23303; Servicebio Ltd) as the secondary antibody for 20 min at 25°C, and visualization was performed using a DAB color development kit (cat. no. G1212-200T; Servicebio Ltd), followed by counterstaining using hematoxylin for 3 min at 25°C. All sections were sealed with neutral resin and scanned with a digital slide scanner (NanoZoomer S20; Hamamatsu Photonics KK). Pathological examination confirmed the diagnosis of poorly differentiated cancer and focal tissue showed liver-like features (Fig. 3). IHC results revealed that the endoscopic biopsy samples were immunopositive for AFP (partial), Hep Par 1 (focal), CK19 (patchy) and CDX2 (homogeneous) (Fig. 4). Based on the elevation in serum AFP and positive AFP staining outcome, the patient was diagnosed with AFPGC. Furthermore, based on the liver-like pathology features, the patient was diagnosed with HAS.
The patient showed no obvious GI-related symptoms throughout the whole observation period before surgery. The patient was then referred to Jiangsu Provincial People's Hospital (Nanjing, China) for radical gastric cancer surgery and the discharge record for this hospitalization was obtained from the patient through a follow-up visit. The patient underwent a 3D laparoscopic-assisted radical total gastrectomy and esophagojejunal Roux-en-Y anastomosis in January 2024 (day 56). The postoperative pathologic resection specimen revealed a lesion located in the lesser curvature of the cardia with a lesion size of 6.5×6.0×2.0 cm, which was styled as Borrmann III in vision and low-differentiated (G3) diffuse tubular adenocarcinoma [International Classification of Diseases for Oncology type 8211/3 (30); T3N0M0 (31)] in the histology. The lesion infiltrated the sub-plasma layer, cancer emboli were seen in the vasculature, there was no clear invasion of nerves, no cancer metastasis in the lymph nodes, and no cancer involvement in the fatty-fibrous connective tissue (data not shown). IHC results revealed that the postoperative resection specimen was immunopositive for human epiderminal growth gator receptor 2 (2+), Ki67 (90%+), postmeiotic segregation increased 2 (partial), MutL homolog 1, MutS homolog (MSH)2, MSH6, AFP, Sal-like protein 4 (partial) and Glypican-3, and negative for in situ hybridization Epstein-Barr encoding region and p53 (data not shown). Post-surgery, the serum AFP level reduced to the normal range (Fig. 5).
The patient was followed up with remotely by telephone in April 2024 (day 148). At the time of this follow-up, the patient was alive and in good postoperative condition.
Discussion
Since the first report in 1970, the definition of AFPGC has remained ambiguous. In the original case reported by Bourreille et al (2), AFPGC was initially defined as GC with excessive serum AFP levels. With the further development of IHC techniques, certain researchers preferred to redefine AFPGC as one type of GC with positive IHC staining for AFP (32). To resolve this divergence, recent guidelines have suggested that AFPGC be defined as GC with both elevated serum AFP levels and positive IHC staining for AFP (33). However, a recent case reported, in which there were GC-related serum AFP elevation and negative IHC staining for AFP, has challenged the definitions from the guidelines (34).
In the present case, as the pathological findings revealed hepatoid features, there was another related disease to discuss. HAS is another rare but aggressive GC-related disease; HAS was first reported by Ishikura et al (24,25) in the 1980s after the observation of certain AFPGC cases with hepatoid features. Subsequent studies have reported that AFP expression is not necessarily observed in certain cases of HAS, further relaxing the definition of HAS as GC with foci of hepatocellular differentiation (Fig. 6) (18,35).
Due to the many histological overlaps between AFPGC and HAS, this ambiguous space of definition often confuses the two, and this confusion has already led to their misuse in certain cases (36). Indeed, it is essential to further clarify the distinction between AFPGC and HAS. During fetal development, AFP is rationally synthesized not only by the liver but also by the yolk sac and gastrointestinal tract. Therefore, the tissues producing AFP in GC can also have different morphologies. Motoyama et al (37) first advocated the typing of AFPGC based on histological features. They proposed three subtypes: i) Hepatoid type; ii) yolk sac tumor-like type; and iii) fetal gastrointestinal type, which has also been referred to as enteroblastic type in classifications by Kinjo et al (16). This classification further highlights the differences and connections between AFPGC and HAS. After finding certain cases that showed only features of common adenocarcinoma, Kinjo et al (16) proposed a fourth type, the common adenocarcinoma type, in 2012. These four subtypes can appear alone or in combination. Recently, a rare case of adenocarcinoma coexisting with squamous cell carcinoma has been reported (34). This may require an expansion of the existing classification.
The diagnosis of AFPGC is extremely heterogeneous and a single diagnosis of only AFPGC may result in a loss of critical information. Certain researchers have tried to eliminate the concept of AFPGC and advocated for a diagnostic system based on histological criteria instead (38), and this viewpoint was supported by the fifth edition of the World Health Organization (WHO) classification of tumors, which includes only HAS and not AFPGC (30).
However, it should be noted that the distinction between AFP-positive and -negative types remains critical when identifying histological type. Chen et al (39) revealed that a group with higher AFP expression had a higher frequency of liver metastasis and worse overall survival compared with those with lower AFP expression among patients with HAS. Moreover, a large-scale epidemiological study has reported the importance of serum AFP levels in patients with GC (40). Therefore, the present report proposes a new classification method to take both histological and AFP-producing features into account. GCs with an elevation in serum AFP levels are defined as serum-positive, and those with positive results in IHC staining for AFP in pathological tissues as tissue-positive. Either serum-positive or tissue-positive should be considered as AFP-positive, and specific serum and IHC manifestations should be marked in the diagnosis. The criteria for serum AFP positivity are widely defined by different values in different studies. Generally, a serum AFP level of >20 ng/ml can be considered as clinically significant and serum AFP positive (40,41). However, certain researchers preferred 100 ng/ml as the cutoff (21,39,42). Nevertheless, there is insufficient evidence for any of the two cutoffs. Considering both cutoffs may be a better option to avoid including cases with physiological variations or focusing too late on potential cases. In many studies, a cutoff value of 300 ng/ml is also used to distinguish very high levels of AFP from others (41,43). Therefore, 20, 100 and 300 ng/ml could be used as cutoffs to grade serum AFP levels, which may be helpful to include more cases previously missed and eliminate the confusion introduced by different definitions of AFP positive cases (Table I). Similarly, a grading system to assess the IHC staining results of AFP is also proposed (Table II). The four-level grading method based on staining intensity is a novel, simple and reproducible method widely used to evaluate or interpret the IHC staining results of many indicators (44–47).
Under the aforementioned definition of AFP positivity, the concepts of AFPGC and HAS were combined to introduce three related new diagnoses: i) AFP-positive hepatoid adenocarcinoma of the stomach (AFPHAS); ii) AFP-positive non-hepatoid adenocarcinoma of the stomach (AFPNAS); and iii) AFP-negative hepatoid adenocarcinoma of the stomach (AFNHAS). This new diagnostic classification would be able to cover all cases encompassed by AFPGC and HAS under all definition methods and establish a fixed definition for each case. This may help prevent confusion and data misapplication in statistical analysis arising from similar cases being classified under different diagnoses with different definition methods.
Furthermore, if further evidence shows that other histological types significantly differ in prognosis or other aspects, AFPNAS may be further categorized into more specific diagnosis groups, including but not limited to AFP-positive yolk-sac-tumor-like adenocarcinoma of the stomach, AFP-positive fetal-gastrointestinal adenocarcinoma of the stomach and AFP-positive common adenocarcinoma of the stomach. AFP-negative cases with other histological types can also be categorized as AFP-negative yolk-sac-tumor-like adenocarcinoma of the stomach, AFP-negative fetal-gastrointestinal adenocarcinoma of the stomach and AFP-negative common adenocarcinoma of the stomach. If needed, AFP-positive squamous-cell carcinoma of the stomach and AFP-negative squamous-cell carcinoma of the stomach can also be discussed.
In the present case, the patient was finally diagnosed as AFPHAS (S3+T3+) according to the proposed new classification system. Several existing meta-analyses (48,49) have conducted preliminary statistical evaluations of various pathologic features, serum AFP levels and survival rates in patients. It is anticipated that a new classification could be further implemented into these data to reveal the impact of serum AFP grading and newly proposed subtypes on patient prognosis. Unfortunately, due to the paucity of literature focusing on differences in the degree of AFP staining in tissues, assessing the impact of tissue AFP grading on patient prognosis remains a task of future work to consider after the present report.
Generally, existing published data is limited by small sample sizes in individual studies and a lack of AFP-level grading. Therefore, whilst these studies (40,41,50) suggest that AFP levels and pathological type impact prognosis, the exact extent of this impact remains unclear. The new classification system proposed in the present report could enable researchers to further categorize both reported and unreported cases, which will allow the exploration of the specific influence of AFP levels and pathological type on survival expectations in patients with GC in more depth. Ultimately, this could help clinicians refine a more precise assessment of the survival expectations of patients, and possibly influence future decisions regarding additional postoperative treatment options.
There is often a genetic factor in the development of cancer, and this may be true for AFPGC cases as well. A whole-exome sequencing study (51) revealed the association between certain genes and AFPGC that AFPGC cases with mutations of multiple genes such as cyclin E (CCNE) 1, Cyclin D1, EGFR, Erb-b2 receptor tyrosine kinase (ERBB) 2, ERBB3, ERBB4, Aurora kinase A, AXL receptor tyrosine, B-cell lymphoma 6, breast cancer gene 2, vascular endothelial growth factor receptor 1, fibroblast growth factor receptor 2, cellular myelocytomatosis oncogene and myeloid cell leukemia 1, tend to have a worse OS rate and exhibit more aggressive behavior than normal GC subtypes, due to the activation of core signal pathways such as RTK/RAS/PI(3)K, p53/cell cycle and JAK/STAT. Based on these findings, CCNE1- and ERBB2-targeted medications may have potential in future AFPGC therapy, which may help to offer differentiated options for patients with AFPGC beyond conventional GC therapies. Unfortunately, the study (51) only used tissue IHC positivity as the inclusion criterion, it did not differentiate between histological types and it omitted serum-positive-only cases. With the new proposed classification system in the present report, researchers in the future could further refine the relationships between mutations, pathways and pathological typing.
Moreover, the patient in the present case was infected with H. pylori and had a history of T2DM. H. pylori is categorized as a group 1 carcinogen by the WHO and is now widely recognized as a primary risk factor for GC (52–55). It is not fully elucidated how H. pylori causes GC, but studies have revealed that the mechanism is related to multiple virulence factors, including cytotoxin-associated gene A, vacuolating cytotoxin A and outer membrane proteins (56,57). Only one previous study explored whether H. pylori serves a different role in different GC subtypes, and this study failed to demonstrate an association between the H. pylori-positive rate and the presence of AFP-positive or hepatoid features in patients with GC (58). Furthermore, compared with H. pylori, the effect of T2DM on the development of GC does not seem to be precise. Certain earlier studies reported an increased risk of GC with pre-existing T2DM (59–61). However, recent research highlight that different study types can lead to different conclusions on this issue. Statistical significance between T2DM history and GC risk has not been demonstrated in prospective studies (62). In addition, a later large-scale study reported that T2DM is unrelated to GC overall but may be associated with excess cardia GC risk (63). In the present case, the tumor was located around the cardia of the patient. If the patient had been able to eradicate H. pylori earlier and received more regular glucose management, they may have been not developed GC. This demonstrates the importance of routine screening for H. pylori and long-term regular glucose monitoring in the primary health care system.
In conclusion, the present report describes the case of an elderly patient with AFPGC and HAS with hepatoid features in the tumor and with both elevated serum AFP levels and positive IHC AFP staining. Although the case could have been diagnosed as both AFPGC and HAS using any existing definition, it was found that the current parallelism of conflicting definition methods of AFPGC and HAS was already a barrier to scientific progress. Therefore, the present report proposed a new classification considering both histological and AFP-producing features, and both expressing features in serum and tissue, to eliminate this obstacle, covering all cases encompassed by AFPGC and HAS under all definition methods and establishing a fixed definition for each case. Moreover, there is currently minimal variation in the treatment approaches for most types of GC, including AFPGC and HAS. Nonetheless, it is crucial to recognize that both AFP level (41) and pathological type (50) serve as independent prognostic factors for GC. The new classification proposed in the present report not only holds significance for pathologists but also extends its relevance to other clinicians by highlighting the importance of AFP levels and pathological type in GC cases. This dual focus is anticipated to remarkably enhance the precision of survival prognosis evaluations. Furthermore, it carries the potential to impact subsequent choices concerning adjunctive postoperative treatments, thereby broadening the scope of personalized patient care strategies in gastric oncological settings. However, further studies are required to elucidate the associations between each specific subtype and genetic-environmental factors such as genes, H. pylori infection and chronic health history, under the new classification method proposed.